In Vitro Analyte Sensor and Methods of Use

ABSTRACT

In vitro electrochemical sensors that provide accurate and repeatable analysis of a sample of biological fluid are provided. Embodiments include sensors that include a sample chambers having overhangs extending therefrom.

FIELD OF THE INVENTION

This invention relates to analytical sensors for the detection ofanalyte.

BACKGROUND OF THE INVENTION

Electrochemical analytical sensors are commonly used to determine thepresence and concentration of a biological analyte. Such sensors areused, for example, to monitor blood glucose levels in diabetic patients.

Although many currently available sensor strip products requirerelatively large sample volumes, e.g., generally requiring 3 μL or moreof blood or other biological fluid, there has been a trend for smallvolume sizes, such as 1 μL and less. For example, U.S. Pat. Nos.6,143,164, 6,338,790 and 6,616,819 provide various configurations ofsmall volume (i.e., less than 1 μL) sensors.

As the volume of sample chambers in the sensors decreases, it becomesincreasingly more difficult to fill the sample chamber with the sampleto be analyzed.

An attempt has been made, in U.S. Pat. No. 6,071,391, to provide anelectrochemical sensor strip that easily receives the sample to beanalyzed. The sample chamber is designed to be partially open at theperipheral part and partially closed by an internal adhesive layer.

As electrochemical sensors continue to be used, there continues to be aninterest in electrochemical sensors that utilize a small sample volumeof biological fluid for analysis and which are easy to fill with sample.

SUMMARY OF THE INVENTION

The electrochemical sensors of the present invention have aconfiguration that facilitates the filling of a sample chamber,particularly a small volume sample chamber. In some embodiments, thesample is maintained in a non-flowing manner in the sample chamberduring analysis.

The sample chamber may be any suitable size, including large and smallvolume sample chambers. In certain embodiments, the sample chamber issized to contain no more than about 1 μL (microliter) of sample, in someembodiments no more than about 0.5 μL, in some embodiments no more thanabout 0.25 μL, and in other embodiments no more than about 0.1 μL ofsample, where in certain embodiments the sample chamber has a volume ofno more than about 0.05 μL or even about 0.03 μL. A measurement zone ispresent within the sample chamber. The measurement zone may have thesame volume, or less volume, than the sample chamber. The sample chambermay be substantially unbounded. For example, a percentage of the samplechamber perimeter may be unbounded, e.g., about 10% or more of theperimeter may be unbounded, e.g., about 50% or more of the perimeter maybe unbounded, e.g., 70% or more, e.g., 80% or more, e.g., 90% or more,e.g., 95% or more of the perimeter may be unbounded. In certainembodiments, the sample chamber may be include linear sides and may beopen to the atmosphere on at least one, two or more linear sides, e.g.,three linear sides. For example, rectangularly shaped sample chambershave six linear sides and at least two or three of which may beunbounded and thus open to the outside environment. Having multiplesides open facilitates filling of the sample chamber with the sample tobe analyzed. Capillary forces pull or otherwise facilitate filing of thesample chamber.

The sensors of the present invention are used for the detection andquantification of an analyte, for example glucose, where in manyembodiments the detection and quantification is accomplished with asmall volume, e.g., submicroliter sample. In general, the invention is asensor for analysis of an analyte in an amount, e.g., small volume, ofsample by, for example, coulometry, amperometry, potentiometry or anycombination thereof. The sensors may also be suitable for use withphotometry.

A sensor of the invention may utilize a non-leachable or non-diffusibleor leachable or diffusible electron transfer agent, such as an enzyme.In many instances, the sensor may additionally or alternately utilize anon-leachable or non-diffusible or leachable or diffusible secondelectron transfer agent, such as a mediator, which can be a redoxmediator.

Sensors of the present invention may include two substrates forming theoverall sensor construction, a spacer between the substrates, a workingelectrode and at least one counter electrode. Together, the twosubstrates and spacer define a sample chamber between the substrates. Atleast a portion of the working electrode and counter electrode arepresent in the sample chamber. In accordance with embodiments of theinvention, the substrates do not align at the sample receiving end oredge of the sensor; rather, at least one of the substrates extends pastthe end of the spacer. In other words, at least one substratecantilevers out past the spacer. In some embodiments, both substratesextend past the end of the spacer, and, for example, one substrate mayextend further than the other substrate. In many embodiments, the sensormay be in the shape of a strip or the like. The substrate overhang orcantilever is, in most embodiments, on the sample receiving end, side oredge of the sensor having the inlet to the sample chamber. In someembodiments, such as tip-filled sensor strips, the spacer cantilever isat the tip of the sensor strip.

These and various other features which characterize the invention arepointed out with particularity in the attached claims. For a betterunderstanding of the sensors of the invention, their advantages, theiruse and objectives obtained by their use, reference should be made tothe drawings and to the accompanying description, in which there isillustrated and described preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the drawings, wherein like reference numerals andletters indicate corresponding structure throughout the several views:

FIG. 1 is a schematic, perspective view of a first embodiment of anelectrochemical sensor strip in accordance with the principles of thepresent invention;

FIG. 2 is a side view of the sensor strip of FIG. 1;

FIG. 3 is an enlarged view of a first end of the sensor strip of FIGS. 1and 2;

FIG. 4 is a top view of a first embodiment of an electrode configurationfor a sensor strip according to the invention, the sensor stripillustrated disassembled, configured to have a working electrode and twocounter electrodes facing each other when assembled;

FIG. 5 is a top view of a second embodiment of an electrodeconfiguration for a sensor strip according to the invention, the sensorstrip illustrated disassembled, configured to have a working electrodeand two counter electrodes facing each other when assembled;

FIG. 6 is a top view of a third embodiment of an electrode configurationfor a sensor strip according to the invention, the sensor stripillustrated disassembled, configured to have a working electrode and acounter electrode planar when assembled;

FIG. 7 is a top view of a fourth embodiment of an electrodeconfiguration for a sensor strip according to the invention, the sensorstrip illustrated disassembled, configured to have a working electrodeand a counter electrode planar when assembled;

FIG. 8 is a top view of a fifth embodiment of an electrode configurationfor a sensor strip according to the invention, the sensor stripillustrated disassembled, configured to have a working electrode and acounter electrode planar when assembled;

FIG. 9 is a top view of a sixth embodiment of an electrode configurationfor a sensor strip according to the invention, the sensor stripillustrated disassembled, configured to have a working electrode and acounter electrode planar when assembled;

FIG. 10 is a top view of the sensor strip of FIGS. 1, 2 and 3;

FIG. 11 is a top view of a second embodiment of a sensor strip;

FIG. 12 is a top view of a third embodiment of a sensor strip;

FIG. 13 is a side view of a second embodiment of an electrochemicalsensor strip in accordance with the principles of the present invention,similar to the first embodiment;

FIGS. 14A, 14B and 14C are schematic illustrations of a process forfilling a sensor strip of FIGS. 1, 2, 3 and 10 with biological fluid;and

FIGS. 15A, 15B and 15C are schematic illustrations of an alternateprocess for filling a sensor strip of FIGS. 1, 2, 3 and 10 withbiological fluid.

DETAILED DESCRIPTION

When used herein, the following definitions define the stated term:

A “biological fluid” is any body fluid in which the analyte can bemeasured, for example, blood, interstitial fluid, dermal fluid, sweat,tears, and urine. “Blood” includes whole blood and its cell-freecomponents, such as, plasma and serum.

A “counter electrode” refers to an electrode, used in conjunction with aworking electrode, through which passes an electrochemical current equalin magnitude and opposite in sign to the current passed through theworking electrode. The term “counter electrode” is meant to includecounter electrodes which also function as reference electrodes (i.e. acounter/reference electrode) unless the description provides that a“counter electrode” excludes a reference or counter/reference electrode.

An “electrochemical sensor” or “electrochemical sensor strip”, andvariations thereof, is a device configured to detect the presence ofand/or measure the concentration of an analyte via electrochemicaloxidation and reduction reactions. These reactions are transduced to anelectrical signal that can be correlated to an amount or concentrationof analyte.

“Electrolysis” is the electrooxidation or electroreduction of a compoundeither directly at an electrode or via one or more electron transferagents (e.g., redox mediators and/or enzymes).

An “electron transfer agent” is a molecule that carries electronsbetween either a redox mediator and the analyte or the working electrodeand the analyte. An electron transfer agent may be used in combinationwith a redox mediator.

The term “facing electrodes” refers to a configuration of the workingand counter electrodes in which the working surface of the workingelectrode is disposed in approximate opposition to a surface of thecounter electrode.

An “indicator electrode” includes one or more electrodes that detectpartial or complete filling of a sample chamber and/or measurement zone.

A “layer” includes one or more layers.

The “measurement zone” is defined herein as a region of the samplechamber sized to contain only that portion of the sample that is to beinterrogated during an analyte assay.

A “non-diffusible,” “non-leachable,” or “non-releasable” compound is acompound which does not substantially diffuse away from the workingsurface of the working electrode for the duration of the analyte assay.

The term “planar electrodes” of “co-planar electrodes” refers to aconfiguration of the working and counter electrodes in which the workingsurface of the working electrode is disposed at least approximatelyplanar to a surface of the counter electrode. “Planar electrodes” or“co-planar electrodes” are typically located on the same substrate.

A “redox mediator” is an agent for carrying electrons between theanalyte and the working electrode, either directly, or via an electrontransfer agent.

A “reference electrode” includes a reference electrode that alsofunctions as a counter electrode (i.e., a counter/reference electrode)unless the description provides that a “reference electrode” excludes acounter/reference electrode.

A “working electrode” is an electrode at which analyte iselectrooxidized or electroreduced with or without the agency of a redoxmediator.

A “working surface” is the portion of a working electrode that iscovered with non-leachable redox mediator and exposed to the sample, or,if the redox mediator is diffusible, a “working surface” is the portionof the working electrode that is exposed to the sample.

“Overhang”’ is the portion of a substrate that extends past or beyondanother substrate. In many embodiments, the substrate that includes anoverhang is a top substrate and the overhang is a longitudinallyextending portion of the substrate.

The in vitro analyte sensors of the present invention may be designed tomeasure the concentration of an analyte in any volume of sample, but areparticularly useful in the determination of analyte concentration in asmall volume of sample, e.g., a sample having a volume no more thanabout 1 μL, for example no more than about 0.5 μL, for example no morethan about 0.25 μL, and further for example no more than about 0.1 μL.In some embodiments, the volume of sample may be as low as 0.05 μL, oras low as 0.03 μL. In some embodiments the biological fluid is blood,and the analyte of interest is glucose or lactate.

As summarized above, the sensors include two substrates separated by aspacer and in many embodiments the sensor has a cantileverconfiguration. The substrates, together with the spacer, form a samplechamber defined by the opposing surfaces of the top and bottomsubstrates and a front surface of the spacer (as will be describedbelow). In many embodiments, one of the substrates (e.g., the topsubstrate) provides an overhang at the sample chamber end (samplefilling end) of the substrate. This overhang provides an extension ofthe space that leads to the sample chamber, which may be a capillaryspace. The overhang may be provided by substrates of unequal lengthsand/or substrates that are displaced along their longitudinal axes. Incertain embodiments, the sample chamber is a capillary chamber and theoverhang provides a capillary extension thereof to facilitate filling ofthe chamber by capillary action. The capillary chamber has a sizesuitable for filling by capillary action, and, in some embodiments, hasonly a single access port. That is, liquid sample ingress and gas (e.g.,air) vent or egress are both via the same access port. An access port,whether for sample ingress, gas egress, or both, may have side that arecurved or otherwise have a radius, or may be linear, e.g., having two ormore (e.g., three) sides of a rectangle.

Referring to the Drawings in general and FIGS. 1-3 and 8 in particular,a first embodiment of an in vitro electrochemical sensor 10 of theinvention is schematically illustrated, and which in this particularembodiment is a small volume sensor. Sensor 10 has a first substrate 12,a second substrate 14, and a spacer 15 positioned therebetween. As willbe described below, sensor strip 10 includes at least one workingelectrode and at least one counter electrode. Sensor 10 is a layeredconstruction, in this particular embodiment having a generallyrectangular shape, i.e., its length is longer than its width, althoughother shapes are possible as well.

Referring to FIG. 2, first or bottom substrate 12 has a first end 12Aand an opposite second end 12B. Second or top substrate 14 has a firstend 14A and an opposite second end 14B. Spacer 15 has a first or front(sample filling) end 15A and an opposite second end 15B. For thisdisclosure, first ends 12A, 14A, 15A are considered the “distal end” andsecond ends 12B, 14B, 15B are considered the “proximal end”. As will bedescribed in detail below, at the distal end of sensor strip 10,substrate first end 12A and substrate first end 14A are intentionallynot aligned. That is, first substrate end 12A and second substrate end14A are intentionally displaced from one another so that a portion ofthe second substrate extends beyond end 12A of the first substrate inthe direction of the longitudinal axis of the sensor. In this embodimentillustrated, spacer end 15A is also not aligned with either end 12A,14A; that is, spacer end 15A is displaced from both substrate end 12Aand substrate end 14A. As described in greater detail below, thisdisplacement of the substrate(s) relative to each other provides anoverhang 17.

The dimensions of a sensor may vary. In certain embodiments, the overalllength of sensor strip 10 may be no less than about 20 mm and no greaterthan about 50 mm. For example, the length may be between about 30 and 45mm; e.g., about 30 to 40 mm. It is understood, however, that shorter andlonger sensor strips 10 could be made. In certain embodiments, theoverall width of sensor strip 10 may be no less than about 3 mm and nogreater than about 15 mm. For example, the width may be between about 4and 10 mm, about 5 to 8 mm, or about 5 to 6 mm. In one particularexample, sensor strip 10 has a length of about 32 mm and a width ofabout 6 mm. In another particular example, sensor strip 10 has a lengthof about 40 mm and a width of about 5 mm. In yet another particularexample, sensor strip 10 has a length of about 34 mm and a width ofabout 5 mm.

Substrates

As provided above, sensor strip 10 has first and second substrates 12,14, non-conducting, inert substrates which form the overall shape andsize of sensor strip 10. Substrates 12, 14 may be substantially rigid orsubstantially flexible. In certain embodiments, substrates 12, 14 areflexible or deformable. Examples of suitable materials for substrates12, 14 include, but are not limited, to polyester, polyethylene,polycarbonate, polypropylene, nylon, and other “plastics” or polymers.In certain embodiments the substrate material is “Melinex” polyester.Other non-conducting materials may also be used.

In this embodiment illustrated in FIGS. 1 through 3, substrate 14, whenmeasured from first end 14A to second end 14B, is longer than substrate12, from first end 12A to second end 12B, the additional length ofsubstrate 14 providing overhang 17. In many embodiments, the length ofsubstrate 12, from first end 12A to second end 12B, is no less thanabout 8 mm and no greater than about 48 mm, e.g., between about 28 mmand about 38 mm, and for example, may be about 30 to about 35 mm. In oneexample, the length is about 31 mm. In many embodiments, the length ofsubstrate 14, from first end 14A to second end 14B, is no less thanabout 10 mm and no greater than about 50 mm, e.g., the length is betweenabout 30 and about 45 mm. In one example, the length is about 32 mm. Thewidth of substrates 12, 14 may be the same or different, and in manyembodiments will be the same, which may be no less than about 1 mm andno greater than about 15 mm in certain embodiments, e.g., about 2-5 mm.The thickness of substrates 12, 14 may be the same or different and mayvary, wherein certain embodiments the thickness may be at least about0.05 mm and generally no greater than about 3 mm, e.g., between about0.20 and about 2 mm. In certain embodiments the thickness is about 0.25mm.

It is understood that both shorter and longer lengths for either or bothsubstrate 12 and substrate 14 could be used, as can wider and/or thickersubstrates 12, 14.

Spacer Layer

As indicated above, positioned between substrate 12 and substrate 14 isspacer 15. Spacer 15 separates first substrate 12 from second substrate14. Spacer 15 is an inert non-conducting substrate, typically at leastas flexible and deformable (or as rigid) as substrates 12, 14. Incertain embodiments, spacer 15 is an adhesive layer or double-sidedadhesive tape or film. Any adhesive selected for spacer 15 should beselected to prevent or minimize diffusion or the release of materialwhich may interfere with accurate analyte measurement.

The thickness of spacer 15 defines the depth of the sample chamber andmay be dimensioned to provide a sample chamber having a capillaryvolume. In certain embodiments, the thickness of spacer 15 may be atleast about 0.01 mm (10 μm) and no greater than about 1 mm or about 0.5mm. For example, the thickness may be between about 0.02 mm (20 μm) andabout 0.2 mm (200 μm). In one certain embodiment, the thickness is about0.05 mm (50 μm), and about 0.1 mm (100 μm) in another embodiment.

The length of spacer 15 may be less than the length of substrate 12and/or of substrate 14, and/or the spacer and one or both substrates maybe displaced along their longitudinal axes. As will be described indetail below, first end 15A of spacer 15 does not align with first ends12A, 14A, but is recessed. Second end 15B may or may not be aligned witheither second end 12B, 14B. The width of spacer 15 may be the same ordifferent than the widths of the substrates, where in many embodimentsthe width is generally the same as the width of substrate 12 andsubstrate 14.

Substrate Overhang

As mentioned above, in accordance with various embodiments of thepresent invention, second substrate 14 extends past spacer 15 at thedistal end; in particular, first end 14A extends past first end 15A;this length of cantilevered substrate is indicated in FIG. 3 byreference number 16. First substrate 12 extends past spacer 15 at thedistal end; in particular first end 12A extends past first end 15A; thislength of extension is indicated in FIG. 3 by reference number 18.Additionally, second substrate 14 overhangs past first substrate 12 atthe distal end; in particular, first end 14A overhangs or extends pastfirst end 12A. This overhang is represented by reference number 17, andis the length of cantilever 16 less extension 18. The dashed lines areincluded in FIG. 3 to aid in alignment of the various features discussedherein. In the embodiment illustrated, there is no other sensor strip 10structure positioned above or below overhang 17 of substrate 14.

The dimensions of cantilever 16 may vary. For ease of description,cantilever 16 may be characterized with respect to the overall length ofsensor 10. For example, cantilever 16 may range from about 0.05% toabout 50% of the length of sensor 10, e.g., from about 1% to about 20%of the length of sensor 10, e.g., from about 4% to about 10%, e.g.,about 6% of the length of sensor 10, although larger and smalleroverhangs could be used. Cantilever 16 may have a length of, or in otherwords, second substrate 14 may extend past spacer layer 15 by at leastabout 0.25 mm, e.g., at least about 0.5 mm, or e.g., at least about 1mm. In certain embodiments, cantilever 16 may be no more than about 20mm. A cantilever 16 of about 2 mm is one specific example.

The dimensions of extension 18 may vary. For ease of description,extension 18 may be characterized with respect to the overall length ofsensor 10. For example, extension 18 may range from about 0.05% to about50% of the length of sensor 10, e.g., from about 1% to about 20% of thelength of sensor 10, e.g., from about 4% to about 10%, e.g., about 4% ofthe length of sensor 10, although larger and smaller extensions could beused. Extension 18 may have a length of, or in other words, firstsubstrate 14 may extend past spacer layer 15 by, at least about 0.25 mm,e.g., at least about 0.5 mm, or e.g., at least about 1 mm. In certainembodiments, extension 18 may be no more than about 20 mm. An extension18 of about 1 mm is one specific example.

As provided above, the distance second substrate 14 extends past oroverhangs first substrate 12 is referred to herein as overhang 17. Insome embodiments, the length of overhang 17 is zero; that is, the lengthof cantilever 16 is the same as the length of extension 18. In thoseembodiments in which an overhang is present, the dimensions of theoverhang may vary. In certain embodiments, the overhang is dimensionedto provide a capillary chamber, with a skin surface providing a wall ofthe thus-formed overhang capillary chamber, to draw, using capillaryforces, sample into sample chamber 20. Accordingly, in certainembodiments the overhang may be sized to provide a capillary spacehaving capillary volume, defined by a surface of cantilever 16 and anopposing surface such as the skin of the user when positioned inopposition thereto. The capillary volume may range from about 10 nL(0.01 μL) to about 10,000 nL (10 μL), e.g., about 100 nL (0.1 μL) toabout 1000 nL (1 μL), e.g., from about 200 nL (0.2 μL) to about 500 nL(0.5 μL). As shown, the overhang and thus the capillary chamber formedby the overhang are in fluid communication with the sample chamber sothat sample contacted with the overhang flows into the sample chamber.

The exact dimensions of overhang 17 may vary. For ease of description,one way to characterize overhang 17 is with respect to the overalllength of sensor 10. For example, overhang 17 may range from about 0.5%to about 20% of the overall length of strip 10, e.g., 1% to about 10%,e.g., 2% to about 5%, e.g., 3% the length of strip 10. In certainembodiments, overhang 17 may be at least about 0.1 mm, and in someembodiments, at least about 0.25 mm, at least about 0.5 mm, and in someother embodiments, at least about 1 mm. In certain embodiments, overhang17 may be no more than about 10 mm, e.g., no more than about 5 mm, e.g.,1 mm, e.g., about 0.25 mm to about 0.5 mm, where an overhang may beabout 0.1 mm in certain embodiments.

Cantilever 16 may be equal to or greater than extension; that is, theratio of cantilever 16:extension 18 may be at least about 1:1. Incertain embodiments, the ratio of cantilever 16:extension 18 may be atleast about 1.5:1 and may be at least about 2:1. The ratio of cantilever16:extension 18 may be no more than about 10:1 and in some embodiment nomore than about 5:1. In one example, extension 18 is about 1 mm andcantilever 16 is about 2 mm; extension 18 is half of cantilever 16,which is a ratio of cantilever 16:extension 18 of 2:1.

In some embodiments, overhang 17 may be about equal to extension 18;that is the ratio of overhang 17:extension 18 may be about 1:1. In someother embodiments, overhang 17 may be equal to about half, or about 50%,of cantilever 16; that is, the ratio of overhang 17:cantilever 16 may beabout 1:2. In still further embodiments, cantilever 16 my be about twicethat of extension 18 (ratio of about 2:1), thus overhang 17 may be abouthalf of cantilever 16 (ratio of about 1:2) and about equal to extension18 (ratio of about 1:1).

Overhang 17 has a sample contacting surface area (i.e., the surface areaof the surface of the overhang that comes in contact with sample)associated therewith, which is the distance or length of overhang×thewidth of overhang 17 (which in most embodiments is the width ofsubstrate 14 and/or sensor strip 10). In certain embodiments, thesurface area of overhang 17 may be at least about 0.1 mm² and no greaterthan about 1 cm², e.g., about 2 mm² to about 30 mm², e.g., about 5 mm²to about 10 mm². In some embodiments, the surface area of overhang 17may be about 1 mm² to about 5 mm².

In many embodiments, second ends 12B and 14B are generally aligned; thatis, neither end 12B, 14B extends or overhangs the other. If there is anoverhang, it may be no more than about 2 mm in certain embodiments.There are some embodiments where a misalignment or overhang exists atsecond end 12B, 14B. For example, second ends 12B, 14B may include anoverhang, tab, indent, or otherwise be configured for connecting to ameter. For the description of sensor strip 10 herein, and of substrates12, 14, it is assumed that second ends 12B, 14B align. Thus, when thereis discussion that one substrate is longer than another, it is assumedthat second ends 12B, 14B are aligned, so that the opposite ends, ends12A, 14A, are not aligned. Spacer end 15B also is generally aligned withends 12B, 14B.

Sample Chamber

Still referring to FIG. 3 and also to FIG. 10, sensor strip 10 includesa sample chamber 20 for receiving a volume of sample to be analyzed.Sample chamber 20 is configured so that when a sample is provided inchamber 20, the sample is in electrolytic contact with both the workingelectrode and the counter electrode, which allows electrical current toflow between the electrodes to effect the electrolysis (electrooxidationor electroreduction) of the analyte. In the embodiment of FIGS. 3 and 8,sensor strip 10 is configured to receive a sample into sample chamber 20at the distal end of sensor strip 10; this distal end is the samplereceiving end of sensor strip 10.

As noted above, sample chamber 20 is defined, in part, by substrate 12,substrate 14 and by first or distal end 15A of spacer 15. Extension 18is the portion of substrate 12 that defines sample chamber 20 and theportion of cantilever 16 of substrate 14 that is not part of overhang 17also defines sample chamber 20. Accordingly, overhang 17 extends from,and is in communication with, sample chamber 20. As shown, a portion ofthe sample chamber perimeter is open or unbounded, where in certainembodiments a substantial portion is unbounded, equal to or greater thana majority of the perimeter of the sample chamber. For example, apercentage of the sample chamber perimeter may be unbounded, e.g., about10% or more of the perimeter may be unbounded, e.g., about 50% or moreof the perimeter may be unbounded, e.g., 70% or more, e.g., 80% or more,e.g., 90% or more, e.g., 95% or more of the perimeter may be unbounded.

As shown in the Figures, this particular sample chamber may becharacterized as having sides such that three elements, substrate 12,substrate 14 and first end 15A, define three sides of sample chamber 20.At least one other side of sample chamber 20 is open, and in thisembodiment, three other sides of sample chamber 20 are open.

Referring to FIG. 10, a top view of sensor strip 10 is illustrated. Fromthis view, sample chamber 20 has sides 20A, 20B, 20C, 20D. Sides 20A,20C and 20D are open to the atmosphere, that is, they are not bounded.Side 20A is defined as the location where first substrate 12 terminatesat first end 12A. Side 20B is defined by first end 15A of the spacer.Sides 20C, 20D are aligned with the side edges of substrates 12, 14. Itis understood that in other embodiments, sample chambers may be designedthat have, e.g., more sides, less sides, curved sides, or otherwisediffer from sample chamber 20.

Sample chamber 20 has a volume sufficient to receive a sample ofbiological fluid therein. In some embodiments, such as when sensor strip10 is a small volume sensor, sample chamber 20 has a volume that is nomore than about 1 μL, for example no more than about 0.5 μL, and alsofor example, no more than about 0.25 μL. A volume of no more than about0.1 μL is also suitable for sample chamber 20, as are volumes of no morethan about 0.05 μL and no more than about 0.03 μL. Sample chamber 20 hasdimensions that facilitate drawing sample to be analyzed into samplechamber 20 by capillary or other surface tensions forces. In embodimentsthat include spacer 15 between substrates 12, 14, the thickness ofsample chamber 20 is generally the thickness of spacer 15.

A measurement zone is contained within sample chamber 20 and is theregion of the sample chamber that contains only that portion of thesample that is interrogated during the analyte assay. In someembodiments, the measurement zone has a volume that is approximatelyequal to the volume of sample chamber 20. In some embodiments themeasurement zone includes 100% of the sample chamber or less, e.g., 80%or less, e.g., 75% or less. In certain embodiments, the sample chamberis a partial fill sample chamber, as described in co-pending applicationtitled “In vitro Analyte Sensor, and Methods” filed Sep. 12, 2005,attorney docket no. 12008.88US01.

Electrodes

As provided above, sensor strip 10 includes a working electrode and atleast one counter electrode. The counter electrode may be acounter/reference electrode. If multiple counter electrodes are present,one of the counter electrodes will be a counter electrode and one ormore may be reference electrodes. Referring to FIGS. 4 through 9, sixexamples of suitable electrode configurations are illustrated.

Working Electrode

At least one working electrode is positioned on one of first substrate12 or second substrate 14. Referring to FIG. 4, working electrode 22 isillustrated on substrate 12. Working electrode 22 includes a conductivetrace 26 extending to the proximal end, such as for connecting to ameter.

Working electrode 22 can be a layer of conductive material such as gold,carbon, platinum, ruthenium dioxide, palladium, or other non-corroding,conducting material. An example of a suitable conductive epoxy isECCOCOAT CT5079-3 Carbon-Filled Conductive Epoxy Coating (available fromW.R. Grace Company, Woburn, Mass.).

Working electrode 22 may be applied on substrate 12 by any of variousmethods. Electrode 22 may be deposited, such as by vapor deposition orvacuum deposition, sputtered, printed on a flat surface or in anembossed or otherwise recessed surface, transferred from a separatecarrier or liner, etched, or molded. Screen-printing is a suitablemethod for applying working electrode 22, although other methods such aspiezoelectric printing, ink jet printing, laser printing,photolithography, and painting can be used.

The material of working electrode 22 typically has relatively lowelectrical resistance and is typically electrochemically inert over thepotential range of the sensor during operation.

Working electrode 22 is provided in sample chamber 20 for the analysisof analyte, in conjunction with the counter electrode, as will bedescribed below.

Counter Electrode

Sensor strip 10 typically includes at least one counter electrodepositioned within sample chamber 20 on substrate 12 or 14. Referring toFIG. 4, two counter electrodes 24 are illustrated on substrate 14. Eachcounter electrode 24 includes a conductive trace 28 extending to theproximal end, such as for connecting to a meter.

Counter electrode 24 may be constructed in a manner similar to workingelectrode 22. Counter electrode 24 may also be a counter/referenceelectrode. Alternatively, a separate reference electrode may be providedin contact with the sample chamber. Suitable materials for thecounter/reference or reference electrode include Ag/AgCl or Ag/AgBrapplied (e.g., printed) on a non-conducting base material or silverchloride on a silver metal base. The same materials and methods may beused to make counter electrode 24 as are available for constructingworking electrode 22, although different materials and methods may alsobe used. Counter electrode 24 can include a mix of multiple conductingmaterials, such as Ag/AgCl and carbon.

Electrode Configurations

Working electrode 22 and counter electrode 24 may be disposed oppositeto and facing each other to form facing electrodes. Referring to FIG. 4,working electrode 22, specifically working electrode 22A, occupies thesurface of substrate 12 that corresponds to sample chamber 20. Counterelectrodes 24, specifically counter electrodes 24A, 24A′, together,occupy less than the total surface of substrate 14 that corresponds tosample chamber 20. When assembled, working electrode 22A overlaps eachof counter electrodes 24A, 24A′, forming facing electrodes.

Referring to FIG. 5, a second facing electrode configuration isillustrated. Working electrode 22B occupies an area on substrate 12significantly less than that which corresponds to sample chamber 20.Counter electrodes 24B, 24B′, together, occupy significantly less thanthe total surface of substrate 14 that corresponds to sample chamber 20.Working electrode 22B is generally equally spaced between the twocounter electrodes 24B, 24B′ but, when assembled, working electrode 22Bdoes not directly overlap counter electrodes 24B, 24B′.

Referring to FIG. 7, a third facing electrode configuration isillustrated. Working electrode 22D, occupies the surface of substrate 12that corresponds to sample chamber 20. Counter electrodes 24D occupiesthe surface of substrate 14 that corresponds to sample chamber 20. Whenassembled, working electrode 22D overlaps counter electrode 24D, formingfacing electrodes.

Referring to FIG. 8, a fourth facing electrode configuration isillustrated. Working electrode 22E occupies the surface of substrate 12that corresponds to sample chamber 20. Counter electrodes 24E occupiesthe surface of substrate 14 that corresponds to sample chamber 20 andadditional area of substrate 14 outside of sample chamber 20. Whenassembled, working electrode 22E overlaps counter electrode 24E, formingfacing electrodes.

Referring to FIG. 9, a fifth facing electrode configuration isillustrated. Working electrode 22F occupies the surface of substrate 12that corresponds to sample chamber 20. Each of two counter electrodes24F, 24F′ occupy approximately half of the area on substrate 14 thatcorresponds to sample chamber 20. Together, electrodes 24F, 24F′ occupyalmost the total surface of substrate 14 that corresponds to samplechamber 20. Working electrode 22 and counter electrode 24 canalternately be disposed generally planar to one another, such as on thesame substrate, to form co-planar or planar electrodes. Referring toFIG. 6, both working electrode 22C and counter electrode 24C occupy aportion of the surface of substrate 14 that corresponds to samplechamber 20, thus forming co-planar electrodes. In the configurations ofFIG. 6, there is no reference electrode or counter/reference electrode,and there is no structure on substrate 12.

As illustrated in FIGS. 4 through 9, the electrodes may be facing orco-planar, and facing electrodes need not be directly opposing eachother. The electrodes may or may not cover the entire area of substrate12, 14. Furthermore, the electrodes need not be the same size.

It is noted that each of the electrodes illustrated include a conductivetrace connecting the electrode to the proximal end of the sensor; suchtraces are used for connecting the electrode to a meter.

Additionally, although not illustrated in FIGS. 4 through 9, sensorstrip 10 may include a fill indicator electrode, to determine when thesample chamber is sufficiently filled with sample.

Alternative Sensor Designs

Referring to FIGS. 11 and 12, two variations of sensor strip 10 areillustrated as sensor strips 40 and 80. Each of sensor strips 40 and 80has a distal end that is rounded, curved, or otherwise has a radiusassociated therewith. Additionally, sensor strip 80 of FIG. 12 has ataper associated therewith, progressing from the proximal end to thedistal end. Sensor strip 40 includes a sample receiving, distal end 40Adefined by distal end 44A of the second substrate. Distal end 44Aoverhangs first substrate's distal end 42A and spacer's distal end 45A.Sensor strip 80 includes a sample receiving, distal end 80A defined bydistal end 84A of the second substrate. Distal end 84A overhangs firstsubstrate's distal end 82A and spacer's distal end 85A.

Referring to FIG. 13, a sensor strip 100, a variation of sensor strip10, is illustrated. Sensor strip 100 includes a first substrate 102having a distal end 102A and a second substrate 104 having a distal end104A. Positioned between substrates 102, 104 is spacer 105. Sensor strip100 is similar to sensor strip 10 except that distal ends 102A, 104Ahave an angle associated therewith. Sensor strip 100 includes overhang107, which is the overhang of distal end 104A past end 102A. Angleddistal ends 102A, 104A can be formed, for example, by slicing off aportion of sensor strip 10. The angled end of sensor strip 100 mayfacilitate filling of the sample chamber, such as when sensor strip 100is pivoted about its distal end (that is, as distal ends 102A, 104A arelowered or otherwise moved over a biological fluid to be analyzed).

Sensing Chemistry

In addition to working electrode 22, sensing chemistry material(s) maybe provided in sample chamber 20 for the analysis of the analyte.Sensing chemistry material facilitates the transfer of electrons betweenworking electrode 22 and the analyte in the sample. Any suitable sensingchemistry may be used in sensor strip 10; the sensing chemistry mayinclude one or more materials.

The sensing chemistry may be diffusible or leachable, or non-diffusibleor non-leachable. For purposes of discussion herein, the term“diffusible” will be used to represent “diffusible or leachable” and theterm “non-diffusible” will be used to represent “non-diffusible ornon-leachable” and variations thereof. Placement of sensing chemistrycomponents may depend at least in part on whether they are diffusible ornot. For example, both non- diffusible and/or diffusible component(s)may form a sensing layer on working electrode 22. Alternatively, one ormore diffusible components may be present on any surface in samplechamber 20 prior to the introduction of the sample to be analyzed. Asanother example, one or more diffusible component(s) may be placed inthe sample prior to introduction of the sample into sample chamber 20.

Electron Transfer Agent

The sensing chemistry generally includes an electron transfer agent thatfacilitates the transfer of electrons to or from the analyte. Theelectron transfer agent may be diffusible or non-diffusible, and may bepresent on working electrode 22 as a layer. One example of a suitableelectron transfer agent is an enzyme which catalyzes a reaction of theanalyte. For example, a glucose oxidase or glucose dehydrogenase, suchas pyrroloquinoline quinone glucose dehydrogenase (PQQ), is used whenthe analyte is glucose. Other enzymes can be used for other analytes.

The electron transfer agent, whether it is diffusible or not,facilitates a current between working electrode 22 and the analyte andenables the electrochemical analysis of molecules. The agent facilitatesthe transfer electrons between the electrode and the analyte.

Redox Mediator

The sensing chemistry may, additionally to or alternatively to theelectron transfer agent, include a redox mediator. Certain embodimentsuse a redox mediator that is a transition metal compound or complex.Examples of suitable transition metal compounds or complexes includeosmium, ruthenium, iron, and cobalt compounds or complexes. In thesecomplexes, the transition metal is coordinatively bound to one or moreligands, which are typically mono-, di-, tri-, or tetradentate. Theredox mediator can be a polymeric redox mediator, or, a redox polymer(i.e., a polymer having one or more redox species). Examples of suitableredox mediators and redox polymer are disclosed in U.S. Pat. No.6,338,790, for example, and in U.S. Pat. Nos. 6,605,200 and 6,605,201.

If the redox mediator is non-diffusible, then the redox mediator may bedisposed on working electrode 22 as a layer. In an embodiment having aredox mediator and an electron transfer agent, if the redox mediator andelectron transfer agent are both non-leachable, then both components aredisposed on working electrode 22 as individual layers, or combined andapplied as a single layer.

The redox mediator, whether it is diffusible or not, mediates a currentbetween working electrode 22 and the analyte and enables theelectrochemical analysis of molecules which may not be suited for directelectrochemical reaction on an electrode. The mediator functions as anagent to transfer electrons between the electrode and the analyte.

Manufacture of the Sensors

Sensor strips 10, 100, described above, are sandwiched or layeredconstructions having substrates 12, 14 spaced apart, such as by spacer15. Such a construction may be made by laminating the various layerstogether, in any suitable manner. An alternate method for making sensorstrips 10, 100 and other sensors in accordance with the invention, is tomold the sensors.

Molding could include positioning at least two spaced apart electricallyconductive electrodes (e.g., wires) in a mold, and molding a body ofinsulative material around the electrodes, with one end having thereinmeans for receiving a fluid sample. More specifically, molding couldinclude positioning at least two spaced apart electrically conductiveelectrodes (e.g., wires) in a mold, before or after molding, treating atleast one of the electrodes with one or more chemicals to change theelectrical properties of the treated electrode upon contact with a fluidsample, and molding a body of insulative material around the electrodeswith one end having therein means for receiving a fluid sample. The bodycan be molded in multiple pieces, e.g., two pieces, with a body and endcap for attaching to one another after the molding is completed, or in asingle piece.

A sensor may be made by determining a suitable length of an overhang fora sensor and manufacturing the sensor so that it includes such anoverhang. For example, a sensor may be made by positioning electrodes onone or more substrates, the substrates including a first substratehaving a first length and a second substrate having a second length,contacting at least a portion of at least one electrode with sensingreagent(s) and configuring the sensor by positioning a spacer betweenthe two substrates to maintain the substrates in a fixed, layeredorientation relative to each other. The substrates are positioned sothat the additional length of the first sensor resides at the distal orsample receiving end of the sensor.

In some embodiments, whether the substrates are the same length or not,the substrates may be displaced relative to each other along theirlongitudinal axes so that one of the substrates, e.g., the topsubstrate, extends a distance beyond the end of the other (e.g., bottomsubstrate) at the sample receiving end to provide the overhang. Contactpads may be positioned at the proximal end.

Application of the Sensor

A common use for the analyte sensor of the present invention, such assensor strip 10, 100, is for the determination of analyte concentrationin a biological fluid, such as glucose concentration in blood,interstitial fluid, and the like, in a patient or other user. Sensorstrips 10 may be available at pharmacies, hospitals, clinics, fromdoctors, and other sources of medical devices. Multiple sensor strips10, 100 may be packaged together and sold as a single unit; e.g., apackage of 25, 50, or 100 strips.

Sensor strips 10 can be used for an electrochemical assay, or, for aphotometric test. Sensor strips 10 are generally configured for use withan electrical meter, which may be connectable to various electronics. Ameter may be available at generally the same locations as sensor strips10, and sometimes may be packaged together with sensor strips 10, e.g.,as a kit.

Examples of suitable electronics connectable to the meter include a dataprocessing terminal, such as a personal computer (PC), a portablecomputer such as a laptop or a handheld device (e.g., personal digitalassistants (PDAs)), and the like. The electronics are configured fordata communication with the receiver via a wired or a wirelessconnection. Additionally, the electronics may further be connected to adata network (not shown) for storing, retrieving and updating datacorresponding to the detected glucose level of the user.

The various devices connected to the meter may wirelessly communicatewith a server device, e.g., using a common standard such as 802.11 orBluetooth RF protocol, or an IrDA infrared protocol. The server devicecould be another portable device, such as a Personal Digital Assistant(PDA) or notebook computer, or a larger device such as a desktopcomputer, appliance, etc. In some embodiments, the server device doeshave a display, such as a liquid crystal display (LCD), as well as aninput device, such as buttons, a keyboard, mouse or touch-screen. Withsuch an arrangement, the user can control the meter indirectly byinteracting with the user interface(s) of the server device, which inturn interacts with the meter across a wireless link.

The server device can also communicate with another device, such as forsending glucose data from the meter and/or the service device to a datastorage or computer. For example, the service device could send and/orreceive instructions (e.g., an insulin pump protocol) from a health careprovider computer. Examples of such communications include a PDAsynching data with a personal computer (PC), a mobile phonecommunicating over a cellular network with a computer at the other end,or a household appliance communicating with a computer system at aphysician's office.

A lancing device or other mechanism to obtain a sample of biologicalfluid, e.g., blood, from the patient or user may also be available atgenerally the same locations as sensor strips 10 and the meter, andsometimes may be packaged together with sensor strips 10 and/or meter,e.g., as a kit.

Sensor strips 10, 100 are particularly suited for inclusion in an‘integrated device’, i.e., a device which has the sensor and a secondelement, such as a meter or a lancing device, in the device. Othersensor strips, such as those having no overhang 17, but where cantilever16 is generally equal to extension 18, would also be suitable forinclusion in an integrated device. The integrated device may be based onproviding an electrochemical assay or a photometric assay. In someembodiments, sensor strips 10, 100 may be integrated with both a meterand a lancing device. Having multiple elements together in one devicereduces the number of devices needed to obtain an analyte level andfacilitates the sampling process.

For example, embodiments may include a housing that includes one or moreof the subject strips, a skin piercing element and a processor fordetermining the concentration of an analyte in a sample applied to thestrip. A plurality of strips 10, 100 may be retained in a cassette inthe housing interior and, upon actuation by a user, a single strip 10,100 may be dispensed from the cassette so that at least a portionextends out of the housing for use.

Operation of the Sensor Strip

In use, a sample of biological fluid is provided into the sample chamberof the sensor, where the level of analyte is determined. In manyembodiments, it is the level of glucose in blood, interstitial fluid,and the like, that is determined. Also in many embodiments, the sourceof the biological fluid is a drop of blood drawn from a patient, e.g.,after piercing the patient's skin with a lancing device or the like,which may be present in an integrated device, together with the sensorstrip.

Embodiments of the subject methods may include contacting the sensor(e.g., an overhang of the sensor) with the skin of a user to form acapillary space and transferring a volume of fluid from a skin incisionwithin the thus-formed capillary space to the sample chamber of thesensor. Accordingly, bodily fluid may be first contacted with at least aportion of one of the substrates of the sensor (e.g., the overhang of atop substrate) prior to being contacted with the other substrate and/orsample chamber.

FIGS. 14A, 14B and 14C illustrate step-wise the filling of sensor strip10 according to an embodiment of the subject invention, which isfacilitated by capillary action provided by the sensor overhang. In FIG.14A, sensor strip 10 is positioned at an angle above a drop ofbiological fluid such as blood to be analyzed. FIG. 14B illustratessensor strip 10 being pivotally lowered onto the drop of blood, so thatsubstrate 14, and particularly overhang 17, is positioned adjacent,e.g., over, the blood. Sensor strip 10 is pivoted at or close to itsproximal end. In this position, when sensor strip 10 is lowered further,a capillary space having a volume as described above, is formed by theoverhang and the skin. The blood sample will directly contact substrate14 at cantilever 16, particularly at overhang 17. Upon contact with theblood, in FIG. 14C, a portion of the blood sample is drawn into samplechamber 20 due to capillary action, which is a function of the surfacecharacteristics and dimensions of sensor strip 10, particularly, thespace between overhang 17 and the surface on which the blood sits. Thesurface of substrate 14 may be hydrophilic, which facilitates drawingthe blood into sample chamber 20. It is understandable that the largerthe overhang, the easier to fill sample chamber 20.

FIGS. 15A, 15B and 15C illustrate step-wise another embodiment of thesubject methods for filling of sensor strip 10. In FIG. 15A, sensorstrip 10 is positioned generally planar to a drop of bodily fluid, e.g.,blood, interstitial fluid, or the like, to be analyzed; sensor strip 10and the blood are typically on the same supporting surface. FIG. 15Billustrates sensor strip 10 being pushed or otherwise moved into thedrop of blood, so that sample chamber 20 is positioned at a level withthe blood. In this position, when sensor strip 10 is pushed further, theblood sample will directly contact side 20A (see FIG. 10) of samplechamber 20. Upon contact with the blood, in FIG. 15C, a portion of theblood sample is drawn into sample chamber 20 due to capillary actionbetween overhang 17 and the surface on which the blood drop sits; thiscapillary action is a function of the surface characteristics anddimensions of sensor strip 10, particularly overhang 17. The capillarychamber of FIG. 15C formed by the overhang is shown completely filled,as is the sample chamber, for exemplary purposes only and is in no wayintended to limit the scope of the invention. It is to be understoodthat the capillary chamber and/or the sample chamber need not fillcompletely or need not be completely or partially filled simultaneously.The filling of the sample chamber may cause partial to complete emptyingof the sample from the capillary chamber.

In a combination of the two above-described methods, sensor strip 100 ofFIG. 13 may be filled by sliding strip 100 toward the blood sample andthen pivoting strip 100 about or near its distal end, to fill the samplechamber. Blood would be drawn into the sample chamber by capillaryaction.

Sensor strip 10, 100 may be operated with or without applying apotential to electrodes 22, 24. In one embodiment, the electrochemicalreaction occurs spontaneously and a potential need not be appliedbetween working electrode 22 and counter electrode(s) 24. In anotherembodiment, a potential is applied between working electrode 22 andcounter electrode(s) 24. The potential may be constant or not. Themagnitude of the potential is dependent on the redox mediator.

The invention of this disclosure is not directed to the potentialutilized, or lack thereof, in use of sensor strip 10, 100. Detailsregarding potential as related to the sensing chemistry and theelectrodes are discussed, for example, in U.S. Pat. No. 6,338,790.

In any event, before, during or after sample is contacted with thesample chamber, the sensor is coupled to a meter and the concentrationof an analyte in the sample, e.g., glucose, is be determined.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it will be apparent toone of ordinarily skill in the art that many variations andmodifications may be made while remaining within the spirit and scope ofthe invention.

All patents, applications and other references in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All patents, patent applications and otherreferences are herein incorporated by reference to the same extent as ifeach individual patent, application or reference was specifically andindividually incorporated by reference.

1-41. (canceled)
 42. A sensor for determining the concentration of an analyte in a sample, the sensor comprising: a first substrate and a second substrate; a sample chamber positioned between the first and second substrates; a first electrode and a second electrode positioned in the sample chamber; wherein the second substrate extends past the first substrate at a sample receiving end of the sensor, wherein the sample chamber comprises at least two unbounded sides at the sample receiving end of the sensor.
 43. The sensor of claim 42, wherein length of the second substrate is greater than length of the first substrate.
 44. The sensor of claim 42, wherein length of the first substrate is equal to length of the second substrate.
 45. The sensor of claim 42, wherein the sample chamber comprises three unbounded sides.
 46. The sensor of claim 42, wherein about 10% to about 95% of the perimeter of the sample chamber is unbounded.
 47. The sensor of claim 46, wherein about 50% to about 95% of the perimeter of the sample chamber is unbounded.
 48. The sensor of claim 42, wherein the sample chamber has a volume of no more than about 1 microliter.
 49. The sensor of claim 42, wherein the sample chamber has a volume of no more than about 0.5 microliter.
 50. The sensor of claim 42, wherein the sample chamber has a volume of no more than about 0.1 microliter.
 51. The sensor of claim 42, wherein the sensor is a glucose sensor or a ketone body sensor.
 52. A sensor for determining the concentration of an analyte in a sample, the sensor comprising: a first substrate and a second substrate; a sample chamber positioned between the first and second substrates; a first electrode and a second electrode positioned in the sample chamber; wherein the second substrate extends past the first substrate at a sample receiving end of the sensor and is configured to form a capillary chamber at a sample receiving end of the sensor when positioned in opposition to a surface, wherein the sample chamber comprises at least two unbounded sides at the sample receiving end of the sensor.
 53. The sensor of claim 52, wherein the capillary chamber has a volume that ranges from about 10 nL to about 10,000 nL.
 54. The sensor of claim 53, wherein the volume ranges from about 100 nL to about 1000 nL.
 55. A system for determining the concentration of an analyte in a sample, the system comprising: a sensor for determining the concentration of an analyte, the sensor comprising: a first substrate and a second substrate; a sample chamber positioned between the first and second substrates; a first electrode and a second electrode positioned in the sample chamber; wherein the second substrate extends past the first substrate at a sample receiving end of the sensor, wherein the sample chamber comprises at least two unbounded sides at the sample receiving end of the sensor; and a meter.
 56. A method of providing a sample to a sample chamber of an analyte sensor comprising: contacting a sample receiving end of sensor, for determining the concentration of an analyte, with the sample, the sensor comprising: a first substrate and a second substrate; a sample chamber positioned between the first and second substrates; a first electrode and a second electrode positioned in the sample chamber; wherein the second substrate extends past the first substrate at a sample receiving end of the sensor, wherein the sample chamber comprises at least two unbounded sides at the sample receiving end of the sensor.
 57. The method of claim 56, further comprising determining the concentration of an analyte in at least a portion of the sample in the sample chamber.
 58. The method of claim 57, wherein the concentration of the analyte is determined using about 1 microliter of sample or less.
 59. The method of claim 56, wherein the concentration of the analyte is determined using coulometry, amperometry, potentiometry, or photometry.
 60. The method of claim 56, wherein the contacting comprises first contacting the sample with the second substrate extending past the first substrate at a sample receiving end of the sensor and then contacting the first substrate and/or the sample chamber with the sample. 